Loss of the RNA polymerase III repressor MAF1 confers obesity resistance.

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

Intestinal microbiota-derived metabolomic blood plasma markers for prior radiation injury.

PURPOSE: Assessing whole-body radiation injury and absorbed dose is essential for remediation efforts following accidental or deliberate exposure in medical, industrial, military, or terrorist incidents. We hypothesize that variations in specific metabolite concentrations extracted from blood plasma would correlate with whole-body radiation injury and dose. METHODS AND MATERIALS: Groups of C57BL/6 mice (n=12 per group) were exposed to 0, 2, 4, 8, and 10.4 Gy of whole-body gamma radiation. At 24 hours after treatment, all animals were euthanized, and both plasma and liver biopsy samples were obtained, the latter being used to identify a distinct hepatic radiation injury response within plasma. A semiquantitative, untargeted metabolite/lipid profile was developed using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, which identified 354 biochemical compounds. A second set of C57BL/6 mice (n=6 per group) were used to assess a subset of identified plasma markers beyond 24 hours. RESULTS: We identified a cohort of 37 biochemical compounds in plasma that yielded the optimal separation of the irradiated sample groups, with the most correlated metabolites associated with pyrimidine (positively correlated) and tryptophan (negatively correlated) metabolism. The latter were predominantly associated with indole compounds, and there was evidence that these were also correlated between liver and plasma. No evidence of saturation as a function of dose was observed, as has been noted for studies involving metabolite analysis of urine. CONCLUSIONS: Plasma profiling of specific metabolites related to pyrimidine and tryptophan pathways can be used to differentiate whole-body radiation injury and dose response. As the tryptophan-associated indole compounds have their origin in the intestinal microbiome and subsequently the liver, these metabolites particularly represent an attractive marker for radiation injury within blood plasma.

Integrated control of hepatic lipogenesis versus glucose production requires FoxO transcription factors.

Insulin integrates hepatic glucose and lipid metabolism, directing nutrients to storage as glycogen and triglyceride. In type 2 diabetes, levels of the former are low and the latter are exaggerated, posing a pathophysiologic and therapeutic conundrum. A branching model of insulin signalling, with FoxO1 presiding over glucose production and Srebp-1c regulating lipogenesis, provides a potential explanation. Here we illustrate an alternative mechanism that integrates glucose production and lipogenesis under the unifying control of FoxO. Liver-specific ablation of three FoxOs (L-FoxO1,3,4) prevents the induction of glucose-6-phosphatase and the repression of glucokinase during fasting, thus increasing lipogenesis at the expense of glucose production. We document a similar pattern in the early phases of diet-induced insulin resistance, and propose that FoxOs are required to enable the liver to direct nutritionally derived carbons to glucose versus lipid metabolism. Our data underscore the heterogeneity of hepatic insulin resistance during progression from the metabolic syndrome to overt diabetes, and the conceptual challenge of designing therapies that curtail glucose production without promoting hepatic lipid accumulation.

Hepatic insulin receptor deficiency impairs the SREBP-2 response to feeding and statins.

The liver plays a central role in metabolism and mediating insulin action. To dissect the effects of insulin on the liver in vivo, we have studied liver insulin receptor knockout (LIRKO) mice. Because LIRKO livers lack insulin receptors, they are unable to respond to insulin. Surprisingly, the most profound derangement observed in LIRKO livers by microarray analysis is a suppression of the cholesterologenic genes. Sterol regulatory element binding protein (SREBP)-2 promotes cholesterologenic gene transcription, and is inhibited by intracellular cholesterol. LIRKO livers show a slight increase in hepatic cholesterol, a 40% decrease in Srebp-2, and a 50-90% decrease in the cholesterologenic genes at the mRNA and protein levels. In control mice, SREBP-2 and cholesterologenic gene expression are suppressed by fasting and restored by refeeding; in LIRKO mice, this response is abolished. Similarly, the ability of statins to induce Srebp-2 and the cholesterologenic genes is lost in LIRKO livers. In contrast, ezetimibe treatment robustly induces Srepb-2 and its targets in LIRKO livers, raising the possibility that insulin may regulate SREBP-2 indirectly, by altering the accumulation or distribution of cholesterol within the hepatocyte. Taken together, these data indicate that cholesterol synthesis is a key target of insulin action in the liver.

Alteration of de novo glucose production contributes to fasting hypoglycaemia in Fyn deficient mice.

Previous studies have demonstrated that glucose disposal is increased in the Fyn knockout (FynKO) mice due to increased insulin sensitivity. FynKO mice also display fasting hypoglycaemia despite decreased insulin levels, which suggested that hepatic glucose production was unable to compensate for the increased basal glucose utilization. The present study investigates the basis for the reduction in plasma glucose levels and the reduced ability for the liver to produce glucose in response to gluconeogenic substrates. FynKO mice had a 5-fold reduction in phosphoenolpyruvate carboxykinase (PEPCK) gene and protein expression and a marked reduction in pyruvate, pyruvate/lactate-stimulated glucose output. Remarkably, de novo glucose production was also blunted using gluconeogenic substrates that bypass the PEPCK step. Impaired conversion of glycerol to glucose was observed in both glycerol tolerance test and determination of the conversion of (13)C-glycerol to glucose in the fasted state. α-glycerol phosphate levels were reduced but glycerol kinase protein expression levels were not changed. Fructose-driven glucose production was also diminished without alteration of fructokinase expression levels. The normal levels of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate observed in the FynKO liver extracts suggested normal triose kinase function. Fructose-bisphosphate aldolase (aldolase) mRNA or protein levels were normal in the Fyn-deficient livers, however, there was a large reduction in liver fructose-6-phosphate (30-fold) and fructose-1,6-bisphosphate (7-fold) levels as well as a reduction in glucose-6-phosphate (2-fold) levels. These data suggest a mechanistic defect in the allosteric regulation of aldolase activity.

SIRT4 coordinates the balance between lipid synthesis and catabolism by repressing malonyl CoA decarboxylase.

Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis, whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism, leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis.

Regulation of lipogenesis by cyclin-dependent kinase 8-mediated control of SREBP-1.

Altered lipid metabolism underlies several major human diseases, including obesity and type 2 diabetes. However, lipid metabolism pathophysiology remains poorly understood at the molecular level. Insulin is the primary stimulator of hepatic lipogenesis through activation of the SREBP-1c transcription factor. Here we identified cyclin-dependent kinase 8 (CDK8) and its regulatory partner cyclin C (CycC) as negative regulators of the lipogenic pathway in Drosophila, mammalian hepatocytes, and mouse liver. The inhibitory effect of CDK8 and CycC on de novo lipogenesis was mediated through CDK8 phosphorylation of nuclear SREBP-1c at a conserved threonine residue. Phosphorylation by CDK8 enhanced SREBP-1c ubiquitination and protein degradation. Importantly, consistent with the physiologic regulation of lipid biosynthesis, CDK8 and CycC proteins were rapidly downregulated by feeding and insulin, resulting in decreased SREBP-1c phosphorylation. Moreover, overexpression of CycC efficiently suppressed insulin and feeding-induced lipogenic gene expression. Taken together, these results demonstrate that CDK8 and CycC function as evolutionarily conserved components of the insulin signaling pathway in regulating lipid homeostasis.

Peripheral effects of FAAH deficiency on fuel and energy homeostasis: role of dysregulated lysine acetylation.

BACKGROUND: FAAH (fatty acid amide hydrolase), primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA). Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH(-/-) mice. METHODOLOGY/PRINCIPAL FINDINGS: FAAH(-/-) mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry). FAAH(-/-) mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN). Fed state skeletal muscle and liver triglyceride levels was increased 2-3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH(-/-) mice. Dysregulated hepatic FAAH(-/-) lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH(-/-) acetyl-CoA (85%, p<0.01) corresponded to similar increases in citrate levels (45%). Altered FAAH(-/-) mitochondrial malate dehydrogenase (MDH2) acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH(-/-) mice was consistent with a compensating contribution from decreased acetylation of fed FAAH(-/-) aldolase B. Fed FAAH(-/-) alcohol dehydrogenase (ADH) acetylation was also decreased. CONCLUSIONS/SIGNIFICANCE: Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH(-/-) mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver's role in fostering the pre-diabetic state, and may reflect part of the mechanism underlying the hepatic effects of endocannabinoids in alcoholic liver disease mouse models.

The fasted/fed mouse metabolic acetylome: N6-acetylation differences suggest acetylation coordinates organ-specific fuel switching.

The elucidation of extra-nuclear lysine acetylation has been of growing interest, as the cosubstrate for acetylation, acetyl CoA, is at a key metabolic intersection. Our hypothesis was that mitochondrial and cytoplasmic protein acetylation may be part of a fasted/re-fed feedback control system for the regulation of the metabolic network in fuel switching, where acetyl CoA would be provided by fatty acid oxidation, or glycolysis, respectively. To test this, we characterized the mitochondrial and cytoplasmic acetylome in various organs that have a high metabolic rate relative to their mass, and/or switch fuels, under fasted and re-fed conditions (brain, kidney, liver, skeletal muscle, heart muscle, white and brown adipose tissues). Using immunoprecipitation, coupled with LC-MS/MS label free quantification, we show there is a dramatic variation in global quantitative profiles of acetylated proteins from different organs. In total, 733 acetylated peptides from 337 proteins were identified and quantified, out of which 31 acetylated peptides from the metabolic proteins that may play organ-specific roles were analyzed in detail. Results suggest that fasted/re-fed acetylation changes coordinated by organ-specific (de)acetylases in insulin-sensitive versus -insensitive organs may underlie fuel use and switching. Characterization of the tissue-specific acetylome should increase understanding of metabolic conditions wherein normal fuel switching is disrupted, such as in Type II diabetes.

Enhanced energy expenditure, glucose utilization, and insulin sensitivity in VAMP8 null mice.

OBJECTIVE: Previous studies have demonstrated that the VAMP8 protein plays a complex role in the control of granule secretion, transport vesicle trafficking, phagocytosis, and endocytosis. The present study was aimed to investigate the role of VAMP8 in mediating GLUT4 trafficking and therefore insulin action in mice. RESEARCH DESIGN AND METHODS: Physiological parameters were measured using Oxymax indirect calorimetry system in 12-week-old VAMP8 null mice. Dynamic analysis of glucose homeostasis was assessed using euglycemic-hyperinsulinemic clamp coupled with tracer radioactively labeled 2-deoxyglucose. Insulin stimulated GLUT4 protein expressions on muscle cell surface were examined by immunofluorescence microscopy. RESULTS: VAMP8 null mice display reduced adiposity with increased energy expenditure despite normal food intake and reduced spontaneous locomotor activity. In parallel, the VAMP8 null mice also had fasting hypoglycemia (84 ± 11 vs. 115 ± 4) and enhanced glucose tolerance with increased insulin sensitivity due to increases in both basal and insulin-stimulated glucose uptake in skeletal muscle (0.19 ± 0.04 vs. 0.09 ± 0.01 mmol/kg/min during basal, 0.6 ± 0.04 vs. 0.31 ± 0.06 mmol/kg/min during clamp in red-gastrocnemius muscle, P < 0.05). Consistent with a role for VAMP8 in the endocytosis of the insulin-responsive GLUT4, sarcolemma GLUT4 protein levels were increased in both the basal and insulin-stimulated states without any significant change in the total amount of GLUT4 protein or related facilitative glucose transporters present in skeletal muscle, GLUT1, GLUT3, and GLUT11. CONCLUSIONS: These data demonstrate that, in the absence of VAMP8, the relative subcellular distribution of GLUT4 is altered, resulting in increased sarcolemma levels that can account for increased glucose clearance and insulin sensitivity.

Advantages of dynamic "closed loop" stable isotope flux phenotyping over static "open loop" clamps in detecting silent genetic and dietary phenotypes.

In vivo insulin sensitivity can be assessed using "open loop" clamp or "closed loop" methods. Open loop clamp methods are static, and fix plasma glucose independently from plasma insulin. Closed loop methods are dynamic, and assess glucose disposal in response to a stable isotope labeled glucose tolerance test. Using PPARalpha(-/-) mice, open and closed loop assessments of insulin sensitivity/glucose disposal were compared. Indirect calorimetry done for the assessment of diurnal substrate utilization/metabolic flexibility showed that chow fed PPARalpha(-/-) mice had increased glucose utilization during the light (starved) cycle. Euglycemic clamps showed no differences in insulin stimulated glucose disposal, whether for chow or high fat diets, but did show differences in basal glucose clearance for chow fed PPARalpha(-/-) versus SV129J-wt mice. In contrast, the dynamic stable isotope labeled glucose tolerance tests reveal enhanced glucose disposal for PPARalpha(-/-) versus SV129J-wt, for chow and high fat diets. Area under the curve for plasma labeled and unlabeled glucose for PPARalpha(-/-) was approximately 1.7-fold lower, P < 0.01 during the stable isotope labeled glucose tolerance test for both diets. Area under the curve for plasma insulin was 5-fold less for the chow fed SV129J-wt (P < 0.01) but showed no difference on a high fat diet (0.30 +/- 0.1 for SV129J-wt vs. 0.13 +/- 0.10 for PPARalpha(-/-), P = 0.28). This study demonstrates that dynamic stable isotope labeled glucose tolerance test can assess "silent" metabolic phenotypes, not detectable by the static, "open loop", euglycemic or hyperglycemic clamps. Both open loop and closed loop methods may describe different aspects of metabolic inflexibility and insulin sensitivity.

The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha.

SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.

NR4A orphan nuclear receptors are transcriptional regulators of hepatic glucose metabolism.

Hepatic glucose production is crucial for glucose homeostasis, and its dysregulation contributes to the pathogenesis of diabetes. Here, we show that members of the NR4A family of ligand-independent orphan nuclear receptors are downstream mediators of cAMP action in the hormonal control of gluconeogenesis. Hepatic expression of Nur77, Nurr1 and NOR1 is induced by the cAMP axis in response to glucagon and fasting in vivo and is increased in diabetic mice that exhibit elevated gluconeogenesis. Adenoviral expression of Nur77 induces genes involved in gluconeogenesis, stimulates glucose production both in vitro and in vivo, and raises blood glucose levels. Conversely, expression of an inhibitory mutant Nur77 receptor antagonizes gluconeogenic gene expression and lowers blood glucose levels in db/db mice. These results outline a previously unrecognized role for orphan nuclear receptors in the transcriptional control of glucose homeostasis.

Amelogenin interacts with cytokeratin-5 in ameloblasts during enamel growth.

The enamel protein amelogenin binds to GlcNAc (Ravindranath, R. M. H., Moradian-Oldak, R., and Fincham, A.G. (1999) J. Biol. Chem. 274, 2464-2471) and to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654-39661). The GMp motif in the N-terminal region of the cytokeratin 14 of ameloblasts binds to trityrosyl motif peptide (ATMP) of amelogenin (Ravindranath, R. M. H., Tam, W., Bringas, P., Santos, V., and Fincham, A. G. (2001) J. Biol. Chem. 276, 36586 - 36597). K14 (Type I) pairs with K5 (Type II) in basal epithelial cells; GlcNAc-acylated K5 is identified in ameloblasts. Dosimetric analysis showed the binding affinity of amelogenin to K5 and to GlcNAc-acylated-positive control, ovalbumin. The specific binding of [3H]ATMP with K5 or ovalbumin was confirmed by Scatchard analysis. [3H]ATMP failed to bind to K5 after removal of GlcNAc. Blocking K5 with ATMP abrogates the K5-amelogenin interaction. K5 failed to bind to ATMP when the third proline was substituted with threonine, as in some cases of human X-linked amelogenesis imperfecta or when tyrosyl residues were substituted with phenylalanine. Confocal laser scan microscopic observations on ameloblasts during postnatal (PN) growth of the teeth showed that the K5-amelogenin complex migrated from the cytoplasm to the periphery (on PN day 1) and accumulated at the apical region on day 3. Secretion of amelogenin commences from day 1. K5, similar to K14, may play a role of chaperone during secretion of amelogenin. Upon secretion of amelogenin, K5 pairs with K14. Pairing of K5 and K14 commences on day 3 and ends on day 9. The pairing of K5 and K14 marks the end of secretion of amelogenin.